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220-plex microRNA expression profile of a single cell

Abstract

Here we describe a protocol for the detection of the microRNA (miRNA) expression profile of a single cell by stem-looped real-time PCR, which is specific to mature miRNAs. A single cell is first lysed by heat treatment without further purification. Then, 220 known miRNAs are reverse transcribed into corresponding cDNAs by stem-looped primers. This is followed by an initial PCR step to amplify the cDNAs and generate enough material to permit separate multiplex detection. The diluted initial PCR product is used as a template to check individual miRNA expression by real-time PCR. This sensitive technique permits miRNA expression profiling from a single cell, and allows analysis of a few cells from early embryos as well as individual cells (such as stem cells). It can also be used when only nanogram amounts of rare samples are available. The protocol can be completed in 7 d.

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Figure 1: Layout of the 96-well plate.
Figure 2: Titration curves of miR-16 in ES-cell total RNA with (red triangles) and without (blue diamonds) pre-PCR amplification.
Figure 3: Expression profile of 220 mouse miRNAs for whole-cell lysate of 10,000 ES cells and 1,000 pg total RNA purified from ES cells.
Figure 4: Titration curve of synthesized miR-16.
Figure 5: miRNA expression profile of DicerFlox/Flox and Dicer−/− MEF single cells.

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Acknowledgements

We are grateful to N.L. Xu, C. Chen, V. Yeung, N. Straus, K. Livak and N. Miyoshi for their helpful suggestions. The work was supported by grants from the Wellcome Trust and the Biotechnology and Biological Sciences Research Council (BBSRC) to M.A.S.

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Correspondence to Kaiqin Lao or M Azim Surani.

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Tang, F., Hajkova, P., Barton, S. et al. 220-plex microRNA expression profile of a single cell. Nat Protoc 1, 1154–1159 (2006). https://doi.org/10.1038/nprot.2006.161

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